Fenchone Derivatives as a Novel Class of CB2 Selective Ligands: Design, Synthesis, X-ray Structure and Therapeutic Potential

A series of novel cannabinoid-type derivatives were synthesized by the coupling of (1S,4R)-(+) and (1R,4S)-(−)-fenchones with various resorcinols/phenols. The fenchone-resorcinol derivatives were fluorinated using Selectfluor and demethylated using sodium ethanethiolate in dimethylformamide (DMF). The absolute configurations of four compounds were determined by X-ray single crystal diffraction. The fenchone-resorcinol analogs possessed high affinity and selectivity for the CB2 cannabinoid receptor. One of the analogues synthesized, 2-(2′,6′-dimethoxy-4′-(2″-methyloctan-2″-yl)phenyl)-1,3,3-trimethylbicyclo[2.2.1]heptan-2-ol (1d), had a high affinity (Ki = 3.51 nM) and selectivity for the human CB2 receptor (hCB2). In the [35S]GTPγS binding assay, our lead compound was found to be a highly potent and efficacious hCB2 receptor agonist (EC50 = 2.59 nM, E(max) = 89.6%). Two of the fenchone derivatives were found to possess anti-inflammatory and analgesic properties. Molecular-modeling studies elucidated the binding interactions of 1d within the CB2 binding site.


Introduction
Fenchone is a bicyclic monoterpene present in essential oils of plant species [1] and is a component of the volatile oil of fresh and air-dried buds of Cannabis sativa [2]. It exerts anti-inflammatory action in rats as noted in a carrageenan-induced right hind-paw edema model [3]. Being a major constituent of Foeniculum vulgare essential oil, fenchone was shown to have an anti-nociceptive activity in the tail-flick pain mouse model, without inducing motor incoordination [4]. Recent data have demonstrated the protective effects of Lavandula stoechas essential oil, where the principal compound is D-fenchone (29.28%), against diabetes and oxidative stress induced by alloxan treatment in rats. Lavender essential oils also decrease kidney and hepatic injuries through their antioxidant properties and play a major role as hepato-and nephroprotection products [5].
Monoterpenes and 5-substituted resorcinols are widely used for the syntheses of cannabinoids [6]. Many of them modulate the endocannabinoid system (ECS), which is an emerging target for the regulation of inflammation and the immune response [7]. ECS activation occurs either via ligands binding to the cannabinoid receptors 1 (CB1R) Here, we present the synthesis and structural identification of twenty-four novel bicyclic monoterpenoid fenchone derivatives with different alkylresorcinol and alkylphenyl groups. We started with the synthesis of fenchone-alkylresorcinols and fenchone-alkylphenols ( Figure 2, Substitution I) in alignment with the previously reported HU-308 [12], JWH-133 [13] and HU-910 [14] (Figure 1). Next, we explored the effect of fluorination of the aromatic ring in the fenchone-alkylresorcinols (Figure 2, Substitution II) bearing different aliphatic substituents. Then, the fenchone-alkylresorcinols with different alkyl substituents were demethylated ( Figure 2, Substitution III). The compounds were characterized by NMR, GCMS and LC-UV-MS (ESI). 1D and 2D NMR experiments (DEPT, gCOSY, TOCSY, HSQC and HMBC) were used to determine the structure assignment of three different fenchone derivatives. The absolute configurations of four derivatives were determined by single-crystal X-ray diffraction. The binding affinities of the fenchone derivatives at the human CB1R (hCB1R) and CB2R (hCB2R) were assessed. Affinity data (K i values) were used to calculate the selectivity indices of these compounds. These ligands were also examined in the [ 35 S]GTPγS binding assay with the aim of evaluation of their functional activity. To assess the in vivo efficacy of the newly developed chemotypes, two compounds from the most potent series were selected to be tested for their anti-inflammatory and anti-nociceptive properties. In addition, we carried out molecular-modeling studies to know the binding interactions of 1d within the CB2 binding site and compare with the parent CB2 compounds.
Introducing fluorine into such molecules can affect conformation, pKa, intrinsic potency, membrane permeability, metabolic pathways and pharmacokinetic properties [18]. Thereby, fluorinated compounds at the aromatic ring of the fenchone derivatives (1a-d) were synthesized (Scheme 4). They were obtained by the reaction of the fenchone-alkylresorcinol dimethyl ether derivatives with 1-chloromethyl-4-fluoro-1,4-diazonia-bicyclo [2.2.2]octane bis(tetra-fluoroborate) or Selectfluor [19].   The optimal conditions involved the use of the Selectfluor reagent in CH 3 CN at r.t under a nitrogen atmosphere. Selectfluor is one of the most reactive electrophilic fluorination reagents and is safe, nontoxic and easy to handle [20]. However, Selectfluor only worked with the fenchone-alkylresorcinol dimethyl ether derivatives (1a-d) with low yield. Fluorination of the fenchone derivatives with the alkylphenol methyl ether (3a-h) was not successful.
Demethylation of aromatic compounds involves the use of acids; however, fenchone upon treatment with acids undergoes rearrangement [21]. Therefore, the fenchone-alkylresorcinol dimethyl ether derivatives (1a-d) were demethylated with sodium ethanethiolate in DMF. However, only one methoxyl group was demethylated (Scheme 5) [22], and the yield was low. Moreover, when trying to demethylate the fenchone-alkylphenol methyl ether derivatives (3a-h), no reaction occurred, meaning that, in our series of compounds, this reagent works only with dimethoxy derivatives.  In total, 24 novel fenchone-based compounds were synthesized, and we grouped these in such a manner as to illustrate the effects of systematic structural variation.

NMR Analysis
The structures of all compounds were determined by 1 H and 13 C NMR and for the fluorinated compounds, 19 F NMR was done. However, a complete analysis of 1D and 2D NMR spectra was performed for 1b, 1d and 5a. The structures were assigned based on the analysis of 1 H, 13 C, DEPT, gCOSY, TOCSY, HSQC and HMBC NMR [23]. Through NMR analysis, it was possible to determine all the chemical shifts for all the carbons and hydrogens. The 2D HSQC permits to obtain a 2D heteronuclear chemical shift correlation map between directly-bonded 1 H and X-heteronuclei (commonly, 13 C and 15 N). Following a 1 H-13 C-HSQC experiment for 1d ( Figure S1, Supplementary Materials), we saw that Carbon 2 (of the fenchone), connected to a hydroxyl group, did not have any cross peaks with the hydrogens and was shifted downfield (Supplementary Materials).

Description of the Crystal Structures
The crystals of 1b, 1d, 4b and 5d were prepared and determined by single crystal X-ray diffraction. Their crystal data and structure refinement are shown in Table S1. The observed hydrogen bonds are listed in Table S2. The molecular ellipsoids (at 50% probability) are shown in Figures S2 and S3.

Affinity for Cannabinoid Receptors
The compounds were further characterized using a radioligand binding assay to determine their affinities for CB1R and CB2R based on each test compound's ability to displace the radiolabeled CB1R/CB2R agonist CP-55,940 from membranes of cells expressing the human CB1R and CB2R [24]. Inhibition constant values (K i ) from the respective competition binding curves are listed in Table 1 in which HU-308 was included for comparison. As shown in Table 1, the fenchone derivatives showed high selectivity towards hCB2R over hCB1R. In most compounds, the (−) analogues prepared from the (−)-fenchone (1b, 1d, 1f, 2b, 3b, 3f, 3h, 4b, 4d, 5b and 5d) showed higher affinity towards hCB2R than their (+) counterparts prepared from (+)-fenchone (1a, 1c, 1e, 2a, 3a, 3e, 3g, 4a, 4c, 5a and 5c). We also observe that the affinity for the hCB2R can be optimized by varying the length of the side chain at C4 for the fenchone-alkylresorcinol dimethyl ether derivatives (1a-f). Thus, the analogue with one methyl group at C4 (1f) had the least affinity to hCB2R (K i = 233 nM) compared to 1b (with a pentyl group) and 1d (with a dimethylheptyl group). Accordingly, 1a (+ isomer) with a pentyl side chain displaced the binding of [  Other potent compounds carry the dimethylheptyl substituent at C4 , which is typical for synthetic cannabinoids. 1c (+ isomer) and 1d (− isomer) with a dimethylheptyl side chain inhibited binding of [ 3 H]CP-55,940 to hCB2R with a K i value of 56.8 nM and 3.51 nM, respectively. The displacement of [ 3 H]CP-55940 by HU-308, 1b and 1d from specific binding sites in membranes from cells expressing hCB2Rs is shown in Figure 4. The change in the position of the chain from the C4 to C5 (2a and 2b) reduced the affinity towards hCB2R by an order of magnitude (K i = 223 and 73.4 nM, respectively). The presence of only one methoxyl group in the aromatic part (3a-h) reduced the affinity to hCB2R dramatically (Table 1). Introducing a fluorine atom in the aromatic part of 1a-d (4a-d) or demethylating it (5a-d) reduced the binding affinity of 1a-d to the hCB2R by almost one order of magnitude (Table 1). Analysis of the structure-activity relationships (SAR) of all of these analogs revealed several structural features for maintaining CB2R affinity and selectivity, including the stereochemistry of the compounds that should be the (−) derivatives, the presence of a branched lipophilic side chain at C4 , dimethoxy groups in the positions C2 and C6 and no substituents in the aromatic ring ( Figure S4).

Functional Characterization
By using the [ 35 S]GTPγS binding assay [24], we next evaluated the activity (agonism, antagonism and inverse agonism) properties of nine key compounds that showed the highest affinity for the hCB2R. Data are listed in Table 2 in which the CB2R agonist HU-308 is included for comparison. Our testing results show that most compounds stimulate the GTPγS binding to CB2R, indicating that these compounds behaved as potent agonists at the hCB2R.
The (−) compounds with the dimethylheptyl side chains at C4 (1d, 4d and 5d) were highly efficacious with 1d being more potent (EC 50 = 2.59 nM; E (max) = 89.6%) than its monomethoxy (5d) (EC 50 = 14.8 nM; E (max) = 105%) and its fluorinated analogues (4d) (EC 50 = 104 nM; E (max) = 119%). The (−) compounds with the pentyl side chain at C4 (1b, 4b and 5b) were less potent and less efficacious than their dimethylheptyl counterparts (Table 2) ( Figure 5). Compound 2b with a hexyl side chain in C5 instead of C4 weakly stimulated the [ 35 S]GTPγS binding to hCB2R.  The most potent compounds in this assay, 1b, 1d and 5d, appeared to be less potent and efficacious than HU-308 at activating the hCB2R in the [ 35 S]GTPγS binding assay (Table 2). However, the mean EC 50 that it displayed in this assay was not significantly different from the corresponding EC 50 values of HU-308 as indicated by the overlap of the 95% confidence limits. Analysis of the SAR revealed that the structural features requirements for maintaining CB2R agonism are the same as those required for maintaining affinity and selectivity ( Figure S4).

Effect on Inflammation and Hyperalgesia (Pain Sensation)
Preclinical studies have shown that CB2R activation mitigates inflammatory reaction and swelling, indicating that CB2R agonists might be a beneficial target for treating inflammatory pain responses [25]. In this study, we used the zymosan-induced inflammation mouse model [26] to investigate the anti-inflammatory and anti-nociceptive activities of the fenchone derivatives, 1b (the (−) enantiomer with a pentyl side chain at C4 ) and 1d (the (−) enantiomer with a dimethylheptyl side chain at C4 ). The compounds were compared to HU-308 and to a vehicle control group in three inflammation assays: paw swelling, pain sensation in the paw and circulating TNF-α ( Figure 6). The extent of hind paw swelling was determined 6 h following paw injection of 60 µg zymosan together with an ip administration of HU-308, 1b or 1d at two doses. The results revealed that both 1b and 1d have anti-inflammatory activity ( Figure 6). HU-308 was able to significantly reduce swelling at a dose of 25 mg/kg with 20% reduction in swelling ( Figure 6A). However, both compounds, 1b and 1d, were able to significantly reduce swelling at the same concentration with 54% reduction in swelling and significantly exceed HU-308 s effect. More importantly, among the three compounds, only 1d reduced swelling at 15 mg/kg with 30% reduction.
The anti-nociceptive effect was determined by the von Frey monofilament assay, and the higher the paw withdrawal threshold, the higher the anti-nociceptive effect of the drug. The two dosages, 15 and 25 mg/kg of 1d showed strong anti-nociceptive effects after 6 h (p < 0.01) ( Figure 6B). 1d was significantly better than 1b and HU-308 at both doses (15 and 25 mg/kg) in reducing pain sensation. Moreover, the levels of TNF-α were reduced significantly for HU-308 (25 mg/kg, 28% inhibition), 1b (25 mg/kg, 39% inhibition) and 1d (15 and 25 mg/kg with 26% and 40% inhibition, respectively) ( Figure 6C). 1b and 1d were slightly but significantly better than HU-308 in reducing TNF-α at 25 mg/kg and 1d at 15 mg/kg was better than both compounds at the same concentration.

Molecular Modeling
We carried out molecular modeling studies to investigate the binding interactions of 1d within the CB2 binding site and compare with the parent CB2 compounds, HU-308 (+) and its (−) enantiomer, HU-433. For better understanding of the interactions and orientations of ligands in the orthosteric binding pocket, we superimposed all best docked poses of the compounds and colored them differently (Figure 7). The electrostatic potential surface shows the hydrophobic nature of the orthosteric site. In our previous studies [27], we reported that the enantiomers HU-308 (+) and HU-433 (−) adopted quite different poses in the binding site of CB2 receptor. The HU-308 has a constrained binding pose, while HU-433 acquired the extending conformation. 1d (-11.582 Kcal/mol) has the similar stereochemistry of HU-433, and therefore it adopted the extended conformation like HU-433 (-11.848 Kcal/mol) in docking calculation. The interaction of CB2 with 1d is mainly from the hydrophobic and aromatic residues of ECL2, TM2, TM3, TM4, TM5, TM6 and TM7. The bicyclic ring of 1d establishes an extensive hydrophobic interaction network with the residues of extracellular side of the pocket, i.e., F91 2.61 , F94 2.64 , F95 2.65 , F106 3.25 and I110 3.29 while the 1,1-dimethylheptyl chain extends towards a deep pocket and forms hydrophobic interactions with the residues F117 3.36 , W194 5.53 , W258 6.48 , V261 6.51 , L262 6.52 and F281 7. 35 .
The affinity was further enhanced by the cooperative π-π interaction between the 2,6-dimethoxy phenyl ring of 1d and F87 2.57 and F183 ECL2 . The interaction of the ligands with CB2 are shown separately in Figures S5 and S6. The docking scores are shown in Table S3.

Conclusions
In the present study, we report the design and synthesis of a series of a novel class of CB2R selective ligands. These fenchone-resorcinol/phenol derivatives were synthesized and characterized by NMR, GCMS and LC-UV-MS (ESI). The X-ray data reinforced the structural elucidation and assignments of all NMR signals. Furthermore, the effects of different alkylresorcinols/phenols on the binding ability of these derivatives to the hCB1R and hCB2R were explored.
Structure-activity relationship studies (SAR) revealed several structural features for maintaining CB2R affinity and selectivity of these analogs. In particular, the (−)-fenchoneresorcinol compounds with branched lipophilic side chain at C4 , dimethoxy groups in the positions C2 and C6 and no substituents in the aromatic ring had high affinity and selectivity for the hCB2R. These structural features were also important for maintaining hCB2R agonism. Compound 1d possessed high affinity (K i = 3.51 nM) and selectivity for the hCB2R and was highly potent and efficacious hCB2R agonist (EC 50 = 2.59 nM, E (max) = 89.6%).
1d was potent in reducing the zymosan-induced paw swelling, pain responses and TNF-α levels in mice. Molecular-modeling analysis elucidated the binding interactions of 1d within the CB2 binding site compared with the parent CB2 compounds, HU-308 (+) and its (−) enantiomer, HU-433. Our data indicate that the design and the development of new terpenoid-derived cannabinoid-like drugs may play a significant role in human disease treatment. NMR spectra were recorded at 500 MHz (1D and 2D 1 H and 13 C NMR) for 1b, 1d and 5a and at 300 MHz ( 1 H, 13 C and 19 F NMR) for the rest of the compounds using deuterated chloroform (CDCl 3 , δ = 7.26 ppm) with tetramethylsilane (TMS) as an internal standard. Thin-layer chromatography (TLC) was run on silica gel 60F 254 plates (Merck). Column chromatography was performed on silica gel 60 Å (Merck). Compounds were located using a UV lamp at 254 nm. GCMS analyses were performed on an HP GCMS instrument (Model GCD PLUS) with an EI detector and a 30 m methyl silicone column. Optical rotations were measured on a polarimeter (Optical Activity) in a 2.00 dm cell at 25 • C.

Synthesis of Fenchone-Resorcinol/Phenol Methylation of Alkyl Phenols/Resorcinols
Methyl iodide (12 mmol) was added to a solution of alkyl phenol/resorcinol (1.51 mmol) and K 2 CO 3 (12 mmol) in dry DMF (5 mL). After stirring at room temperature for 24 h, the mixture was diluted with water (40 mL) and extracted with ether. The organic layer was washed with water, dried and evaporated followed by purification by column chromatography on silica gel with ether/petroleum ether (2-4%).

Fluorination with Selectflour ®
We dissolved 0.48 mmol of Selectflour ® in 2.6 mL acetonitrile. The solution was cooled to 0-5 • C and fenchone-dimethoxyalkylresorcinol (0.48 mmol) in 2.6 mL acetonitrile was added to it. The reaction was stirred at this temperature for 1.5 h, and then the reaction was left stirring overnight at room temperature. Ether was added to the reaction mixture, and then the mixture was washed with brine. Removal of the solvent afforded the desired products, which were purified by chromatography with ether/petroleum ether (0-2%) [19].

Demethylation with Sodium Ethanethiolate/DMF
We added 4-8 mL (2-4 mmol) of a 0.5 M solution of NaSEt in DMF to the aromatic methoxy compound (1 mmol), and the resulting solution was heated in an oil bath at 115-120 • C under N 2 . The completion of the reaction in each case was determined by TLC. The cooled reaction mixture was then acidified with 10% aqueous HCl and extracted with EtOAc (3 × 10 mL). The combined organic extracts were washed with 10% aqueous NaOH (3 × 3 mL) and H 2 O (3 mL) and dried (MgSO 4 ). Removal of the solvent afforded the desired products, which were purified by chromatography with ether/petroleum ether (6-8%) [22].

X-ray Crystallography
A single crystal of the compound was attached to a 400/50 MicroMeshes™ with NVH Oil [28] and transferred to a Bruker SMART APEX CCD X-ray diffractometer equipped with a graphite-monochromator. The system was controlled by a pentium-based PC running the SMART software package [29]. MoKα radiation (λ = 0.71073 Å) was used for data collection at room temperature, and CrysAlisPro [30] was used for data processing using Olex2 [31]. The SHELXT [32] structure solution program using Intrinsic Phasing was used for structure solving, and the SHELXL [33] refinement package using Least Squares minimization was used for structure refinement.

Binding Assays
Binding to the CB1R was assessed in a competition displacement assays using [ 3 H]CP-55,940 as the radioligand [24]. Membranes from cells expressing hCB1R and hCB2R were purchased from Charles River, (Cat#A317 and A318, respectively; OH, USA). Solutions of test compounds ranging from 0.1 nM to 10 mM were prepared in DMSO. The desired amount of membrane preparation was diluted with ice-cold assay buffer (50 mM Tris-HCl, 2.5 mM EDTA, 5 mM MgCl 2 , 0.1% BSA, pH 7.4) and was vortexed. We distributed 100 µL of compound into each tube, followed by addition of 800 µL of diluted membranes (1 µg/tube) and kept on ice until the addition of

[ 35 S]GTPγS Binding Assay
The method used for measuring agonist-stimulated binding of [ 35 S]GTPγS was based on a described protocol [24]. Membranes (5 µg protein) were incubated in GTPγS assay buffer (50 mM Tris HCl (pH 7.4), 0.2 mM EGTA, 9 mM MgCl 2 , 150 mM NaCl, 1 mg/mL BSA) containing 100 µM GDP, 0.05 nM [ 35 S]GTPγS and test compounds at various concentrations. Vacuum filtration was used to separate bound ligand from free ligand. We used 10 µM GTPS to determine nonspecific binding. Basal binding was assayed in the absence of the ligand and in the presence of GDP.
Control mice were injected with vehicle. Paw swelling and pain perception were measured after 2, 6 and 24 h [26].

Pain Assay
The paw withdrawal von Frey test was used to evaluate hyperalgesia at 2, 6 and 24 h following injections of zymosan and/or the test compounds [26]. In the von Frey nociceptive filament assay, von Frey calibrated monofilament hairs of logarithmically incremental stiffness (1.4-60 g corresponding to 4.17 to 5.88 log of force) was used to test the mouse sensitivity to a mechanical stimulus on the swollen paw. The measurements were done in a quiet room. The animals were held for 10 s prior to paw pain measurements. The investigator applied the filament to the central area of the hind paw with gradual increasing size. The middle of the hind paw was poked to provoke a flexion reflex followed by a clear flinch response after paw withdrawal. Each filament was applied for 3-4 s to induce the end-point reflex. The force filament of 1.4 g was used for the first testing. In the case of no withdrawal response, the next higher stimulus was tried. The mechanical threshold force (in grams (g)) was defined as the lowest force imposed by two von Frey monofilaments of various sizes, required to produce a paw retraction. The untreated left hind paw served as a control.

Measurement of TNFα
After 24 h of zymosan injection, blood was collected, and the sera were assayed for TNFα. A mouse TNFα ELISA kit was used according to the manufacturer's instructions (R&D Systems, Minneapolis, MN, USA).

Molecular Modelling Studies
The three-dimensional structure of human CB2 (PDB ID: 5ZTY) was downloaded from the protein databank. The missing residues between 222 and 235 were modelled, and mutations were reverted to wildtype residues. The protonation states of all acidic and basic residues were assigned at physiological pH 7.2. The retrained minimization considering 0.30 Å root mean square deviation (RMSD) of all atoms was performed using optimized potentials for a liquid simulation extended (OPLS3e) force field.
All docking calculations were performed using two different docking protocols, Schrodinger suit 2020.3 [34] and Autodock 4.5.7 [35]. The orthosteric ligand binding site was defined by generating 20 Å grid around the co-crystallized small molecule (AM10257) in Glide, whereas 60 × 60 × 60 grid points with a 0.375 Å spacing around the centroid of AM10257 were generated in Autodock.
The compounds were prepared at pH 7.0 ± 2.0 using the LigPrep module. The docking calculations were performed using the default protocol of GLIDE module. The 10 conformations of each compound were generated using Standard precision (SP) docking. The 10 poses of each conformation were generated using Extra precision (XP) docking. The best pose was selected based on the lowest energy and interaction with the active site residues. Lamarckian Genetic Algorithm was used in Autodock to identify the binding poses of each compound.
The receptor was kept rigid, whereas the ligand was allowed torsional flexibility. The default parameters were set but with 2.5 × 10 7 energy evaluations. The 50 poses of each compound were generated using the Lamarckian Genetic Algorithm. The resulting poses were clustered into groups of 2.0 Å root-mean-square deviation (rmsd). The best scoring pose from the group with a higher number of conformers was chosen as the final pose. Both software demonstrated similar lowest energy poses of the ligands.

Statistical Analyses
Statistical analysis was performed with GraphPad Prism software. Statistical analysis details are listed under each figure. The results are presented as the value ± SE (standard error). In rare cases where all the measurements give the same values, no SE bar is presented, as no error can be measured. * p < 0.05 comparing to control group. # p-value < 0.05 in the indicated comparison.
Supplementary Materials: The following supporting information are available online: Figure S1: The HSQC of compound 1d; Figure S2: Crystal structures of 1b and 4b; Figure S3: Crystal structures of 1d and 5d; Table S1: Crystal data and structure refinement for 1b, 4b, 1d, and 5d; Table S2: Hydrogen bonds for 1b, 4b, 1d, and 5d (Å, u); Figure S4: Structural Requirements for CB2 affinity and selectivity; Figure S5: Binding site of the CB2 cavity is represented by electrostatic potential surface. The residues of binding site and ligands are represented by thin and thick tubes respectively. H-bonds and π-π interactions are represented by orange and cyan dotted lines respectively. A. 5ZTY_Ligand B. HU-308 C. HU-433 D. 1d; Figure S6: Two dimensional representation of proteinligand interactions. A. 5ZTY_Ligand B. HU-308 C. HU-433 D. 1d; Table S3: Docking Glide XP score with energy decomposition; LC-UV-MS (ESI); NMR data for 1b; NMR data for 1d; NMR data for 5a.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.